Duckweed is widely used in environmental biotechnology and has recently emerged as a potential feedstock for biofuels due to its high growth rate and starch content. The genetic diversity and composition of a natural duckweed population in genera Spirodela, Landoltia and Lemna from Lake Tai, China, were investigated using probabilistic analysis of multilocus sequence typing (MLST). The 78 strains were categorized into five lineages, among which strains representing L. aequinoctialis and S. polyrhiza were predominant. Among the five lineages, interlineage transfers of markers were infrequent and no recombination was statistically detected. Tajima's D tests determined that all loci are subject to population bottlenecks, which is likely one of the main reasons for the low genetic diversity observed within the lineages. Interestingly, strains of L. turionifera are found to contain small admixture from L. minor, providing rare evidence of transfer of genetic materials in duckweed. This was discussed with respect to the hypothesis that a cross of these two gave rise to L. japonica. Moreover, the conventional maximum-likelihood phylogenetic analysis clearly recognized all the species in the three genera with high bootstrap supports. In conclusion, this work offers a basic framework for using MLST to characterize Spirodela, Landoltia and in particular Lemna strains at the species level, and to study population genetics and evolution history of natural duckweed populations.